Comprehensive Identification of the Genetic Requirements for ESX-1 Secretion

描述(由申请人提供)：ESX-1(ESAT-6系统1)分泌系统是分枝杆菌和革兰氏阳性病原体的关键毒力决定因素。由于ESX-1功能所需的所有基因尚未确定，因此对这种分泌系统的分子机制的了解存在着根本性的差距。缺乏这一信息是因为由于在直接、高通量测量ESX-1功能方面的技术限制，从未对ESX-1分泌需求进行饱和遗传筛查。然而，这些知识的获得对于基本了解分枝杆菌的毒力是至关重要的。长期目标是了解作为分枝杆菌致病基础的蛋白质分泌系统的基本分子机制。这项建议的目标是通过鉴定致病分枝杆菌分泌ESX-1所需的所有基因来实现这一目标的初步步骤。假设这可以使用直接饱和的基因筛查来监测ESX-1的功能。一种基于蛋白质组学的方法，允许直接、高通量监测分枝杆菌ESX-1底物的分泌，并在申请人的实验室中得到验证。支持这一提议的理由是，在完成这一项目过程中积累的知识将极大地促进对这一重要毒力途径的理解。因此，提出了以下具体目标：进行直接、饱和的遗传筛查，以全面鉴定致病分枝杆菌ESX-1分泌系统的组成部分。在申请人手中，已经建立了一种使用全菌落MALDI-TOF(基质辅助激光解吸电离飞行时间)质谱仪直接监测细菌中ESX-1蛋白分泌的方法是可行的。该方法将用于对海洋分枝杆菌转座子文库进行大规模筛选，以识别不分泌ESX-1底物的菌落。拟议的办法是创新的，因为它直接脱离现状，并将克服目前在确定ESX-1系统所有部件方面的技术障碍。这一贡献将是重大的，因为它有望导致对ESX-1分泌的基本生物学的理解有很大进步。 公共卫生相关性：拟议的研究与公共卫生相关，因为了解分枝杆菌病原体如何致病的基本生物学是开发新的抗结核病诊断和治疗方法的第一步，这将有助于缓解当前的结核病流行。这项研究与NIH使命的一部分有关，该使命与追求基础知识有关，这将导致开发有助于疾病预防的科学资源。

DESCRIPTION (provided by applicant): The ESX-1 (ESAT-6 system 1) secretion system is a key virulence determinant in both mycobacteria and Gram-positive pathogens. There is a fundamental gap in understanding the molecular mechanisms of this secretion system because all of the genes required for ESX-1 function have not been identified. The absence of this information is because a saturating genetic screen for requirements of ESX-1 secretion has never been performed due to technical limitations in the direct, high throughput measurement of ESX-1 function. However, the acquisition of such knowledge is critical to the basic understanding of mycobacterial virulence. The long-term goal is to understand the basic molecular mechanisms of protein secretion systems that underlie mycobacterial pathogenesis. The objective of this proposal is to attain the primary step toward this goal by identifying all of the genes required for ESX-1 secretion in pathogenic mycobacteria. The hypothesis is that this can be accomplished using a direct saturating genetic screen to monitor ESX-1 function. A proteomics based method that allows direct, high throughput monitoring of secretion of mycobacterial ESX-1 substrates was devised and validated in the applicant's laboratory. The rationale that underlies this proposal is that the knowledge amassed in the completion of this project will greatly advance the understanding of this important virulence pathway. Therefore, the following specific aim has been proposed: To conduct a direct, saturating genetic screen to comprehensively identify the components of the ESX-1 secretion system in pathogenic mycobacteria. A method using whole colony MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry to directly monitor ESX-1 protein secretion in bacteria has been established as feasible in the applicant's hands. This method will be used to conduct large scale screening of a Mycobacterium marinum transposon library to identify colonies that do not secrete ESX-1 substrates. The proposed approach is innovative because it represents a direct departure from the status quo and will overcome the current technical hurdle to identifying all components of the ESX-1 system. This contribution will be significant because it is expected to result in a great advance in understanding the basic biology of ESX-1 secretion. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because understanding the basic biology of how mycobacterial pathogens cause disease is the first step in developing new anti- Tuberculosis diagnostics and therapeutics, which would help alleviate the current Tuberculosis epidemic. This research is relevant to the part of NIH's mission that relates to the pursuit of fundamental knowledge that will lead to the development of scientific resources that will assist in disease prevention.